Alpha 1-antitrypsin is a protease inhibitor present in mammalian blood whose apparently major physiological function is to inhibit elastase, a potent protease which hydrolyzes structural proteins. Alpha 1-antitrypsin also inhibits other serine proteases. The normal plasma level of alpha 1-antitrypsin is about 2 mg/ml. A low level of alpha-1-antitrypsin in the blood may be associated with chronic obstructive pulmonary emphysema and infantile liver cirrhosis. Under many inflammatory conditions, an acute-phase response is initiated and the concentration of alpha-1-antitripsin is substantially increased. In order to study and treat alpha-1-antitrypsin deficiency and to be involved in the mechanism of the acute-phase response, it is therefore desirable to have a pure alpha-1-antitrypsin protein. In particular, it is desirable to have a source of antitrypsin protein produced by microorganisms through genetic engineering techniques.
The sequencing of chromosomal DNA coding for alpha antitrypsin has been described by Kurachi et al., Proc. Natl. Acad. Sci. U.S.A., 78, 6826-6830 (1981) and by Chandra et al., Biochem. Biophys. Res. Comm., 103, 751-758 (1981), the disclosures of which are incorporated herein by reference.
U.S. Pat. Nos. 4,839,282 and 5,218,091 which are incorporated herein by reference, discloses a process for expressing human non-glycosylated alpha 1-antitrypsin in Saccharomyces cerevisiae (Bakers yeast) which is difficult to upscale.
To overcome the major problems associated with the expression of recombinant gene products in S. cerevisiae (e.g., loss of selection for plasmid maintenance and problems concerning plasmid distribution, copy number and stability in fermentors operated at high cell density), a yeast expression system based on methylotrophic yeast, such as for example, Pichia pastoris, has been developed. A key feature of this unique system lies with the promoter employed to drive heterologous gene expression. This promoter, which is derived from a methanol-responsive gene of a methylotrophic yeast, is frequently highly expressed and tightly regulated (see, e.g., European Patent Application No. 85113737.2, published Jun. 4, 1976, under No. 0 183 071 and issued in the U.S. on Aug. 8, 1989, as U.S. Pat. No. 4,855,231). Another key feature of expression systems based on methylotrophic yeast is the ability of expression cassettes to stably integrate into the genome of the methylotrophic yeast host, thus significantly decreasing the chance of vector loss.
Although the methylotrophic yeast P. pastoris has been used successfully for the production of various [Cregg et al., Bio/Technology 5, 479 (1987)], lysozyme and invertase [Digan et al., Developments in Industrial Microbiology 29, 59 (1988); Tschopp et al., Bio/Technology 5, 1305 (1987)], endeavors to produce other glycosylated heterologous gene products in Pichia, especially by secretion, have given mixed results. At the present level of understanding of methylotrophic yeast expression systems, it is unpredictable whether a given gene can be expressed to an appreciable level in such yeast of whether the yeast host will tolerate the presence of the recombinant gene product in its cells. In addition, it is unpredictable whether desired or undesired proteolysis of the primary product will occur, and if the resulting proteolytic products are biologically active. Further, it is especially difficult to foresee if a particular protein will be secreted by the methylotrophic yeast host, and if it is, at what efficiency. Even for the non-methylotrophic yeast S. cerevisiae, which has been considerably more extensively studied than P. pastoris, the mechanism of protein secretion is not well defined and understood.
U.S. Pat. No. 5,612,198 to Brierley et al, which is herein incorporated by reference, discloses the production of insulin-like growth factor-1 in methylotrophic yeast.